The invention relates to methods and medicines for inhibiting blood coagulation.
Blood Coagulation
Blood coagulation is a process whereby blood thickens and gradually becomes a clot. The process is vitally important to the stoppage of bleeding when blood vessels are damaged. Blood coagulation occurs through a complex series of molecular reactions, ultimately resulting in conversion of soluble fibrinogen molecules, present in the blood, into insoluble threads of fibrin. The result is a blood clot which consists of a plug of platelets enmeshed in the insoluble fibrin network.
During the blood coagulation process, a cascade of proteins in the blood, called xe2x80x9cclotting factors,xe2x80x9d are activated and catalyze the chemical reactions that result in a blood clot. These clotting factors comprise two convergent reaction pathways, initiated by different stimuli, both leading to clot formation. Clot formation in response to blood vessel damage results from activation of the extrinsic pathway. See FIG. 1. This pathway is initiated by display of tissue factor (xe2x80x9cTFxe2x80x9d) protein on the surface of damaged blood vessels. Exposed TF binds to circulating factor VIIa to form an active protease that cleaves factor X to active factor Xa.
Blood clot formation in response to abnormalities in the blood vessel wall, in the absence of tissue injury, results from activation of the intrinsic pathway. See FIG. 1. This pathway is initiated by factor XII when contact is made between blood and exposed endothelial cell surfaces. This pathway, in a sequential reaction cascade involving factors XI, IX and VIII, and the active xe2x80x9caxe2x80x9d forms of each of these factors, results in formation of factor Xa from factor X.
The formation of factor Xa from factor X is the point at which the extrinsic and intrinsic pathways converge. See FIG. 1. The resulting factor Xa then binds factor Va to form prothrombinase. Prothrombinase is a protease that cleaves prothrombin (also called xe2x80x9cfactor IIxe2x80x9d) to yield thrombin (also called xe2x80x9cxcex1-thrombinxe2x80x9d or xe2x80x9cfactor Iaxe2x80x9d). xcex1-Thrombin cleaves fibrinogen to form soluble fibrin monomers. xcex1-Thrombin also cleaves factor XIII to active factor XIIIa. Factor XIIIa causes formation of covalent bonds between the soluble fibrin monomers, converting them into an insoluble fibrin polymer meshwork which, when combined with platelets, is the clot.
Inhibition and Reversal of Blood Coagulation The blood coagulation process described above is regulated by an opposing group of factors, called anticoagulants, that inhibit coagulation or blood clotting. For example, formation of the TF/factor VIIa complex essential for progression of the extrinsic pathway, is inhibited by a protein called tissue factor pathway inhibitor (xe2x80x9cTFPIxe2x80x9d). See FIG. 1. Factors Va (interacts with factor Xa) and VIIIa (intrinsic pathway) are inhibited by anticoagulant-activated protein C (xe2x80x9cAPCxe2x80x9d) and its associated cofactor, protein S. Production of APC is activated by a-thrombin after binding to thrombomodulin. Finally, antithrombin III (xe2x80x9cATIIIxe2x80x9d) functions by inactivating factor Xa and thrombin.
At any given time in the blood, the overall balance of blood coagulants and blood anticoagulants determines whether blood will clot. Normally, the balance is in favor of the anticoagulants and the blood circulates freely throughout the body. However, in response to injury or trauma, the coagulants increase in concentration and cause clotting of the blood.
In addition to physiologic inhibitors of blood coagulation, the body possesses a system to actively remove clots that have already formed. Circulating blood contains plasminogen, which binds to the fibrin molecules comprising a blood clot. Nearby cells release an inactive form of tissue plasminogen activator (xe2x80x9cTPAxe2x80x9d) which binds to fibrin, is subsequently activated, then cleaves plasminogen to plasmin. Plasmin digests fibrin and dissolves the clot.
There exist human disorders, called xe2x80x9cthromboses,xe2x80x9d where blood clots when it normally should not. Thrombosis is a major cause of death due to occlusion of arteries, which leads to heart attacks, strokes and peripheral ischemia (i.e., local deficiencies in blood supply). Thrombosis can also cause occlusion of venous blood vessels and result in pulmonary emboli.
In order to prevent or treat such thrombotic disorders, therapeutic methods to inhibit clot formation or to dissolve clots have been developed. Existing anticoagulants (that inhibit blood clot formation), for example, include heparin, which greatly increases activity of the physiologic anticoagulant, ATIII, in the blood. Warfarins are anticoagulants that are vitamin K antagonists. Since vitamin K is required for synthesis or functioning of a number of clotting factors (i.e., factors II, VII, IX and X, as well as a-thrombin and proteins C and S), sequestration of vitamin K inhibits coagulation.
The existing blood anticoagulants, however, produce side effects. For example, heparin administration can cause bleeding and thrombocytopenia (i.e., decrease in platelets). A disadvantage of warfarins is that it takes several days for their maximum effect to be realized. As with heparin, bleeding can also be a complication. Warfarins are also teratogens and can cross the placenta, causing fetal abnormalities when administered to pregnant women.
Thrombolytic agents, which dissolve existing clots, are also used therapeutically. Their activity is based on enhancing the generation of plasmin from its plasminogen precursor. Such agents include recombinant TPA and streptokinase. Disadvantages of these thrombolytics include a systemic fibrinolytic activity that can result in bleeding throughout the body. Some thrombolytics (i.e., streptokinase) are also highly antigenic and can cause allergic reactions.
Therefore, there are problematic side effects with existing anticoagulant and thrombolytic drugs. An ideal drug that prevents blood clot formation would target single clotting factors such that side effects resulting from nonspecific action of the drug are eliminated. Such ideal drugs would have superior efficacy and safety profiles since thromboses would be inhibited without bleeding as a side effect. Additionally, because of the many different manifestations and etiologies of thrombosis, and the different locations in the body where clots can form, there is a need for new and varied treatments for these manifestations.
In accordance with the present invention, it has been discovered that certain peptides derived from the region comprising amino acids 307 to 356 of the human blood coagulation factor Va exhibit excellent blood anticoagulation properties with little or no adverse side effects.
Thus, the present invention provides, as a new composition of matter, a peptide which is from 3 to 50 amino acids in length which contains a sequence (a) of at least 3 amino acids which is identical to a sequence found within amino acids 307 to 356 of the human blood clotting factor Va (SEQ ID NO. 1), but excluding the peptide comprising amino acids 311 to 325 (SEQ ID NO. 15) and the peptide comprising amino acids 321 to 335 (SEQ ID NO. 16). The peptide can further contain additional amino acid sequences at the N-terminal end of sequence (a), the C-terminal end of sequence (a), or both the N- and C-terminal ends of sequence (a), as long as the amino acid sequence of the entire peptide is at least 60% identical to a sequence within SEQ ID NO. 1. The peptide further exhibits an IC50 of between 50 nM to 500 xcexcM for inhibition of prothrombinase
In addition, the present invention also provides a pharmaceutical composition comprising one or more of the above inhibitory peptides and a pharmaceutically acceptable carrier.
The present invention also provides new processes for reducing and preventing unwanted clotting of blood in mammals, including humans, comprising administering these pharmaceutical compositions to the mammals.